This proposal seeks continued support for exploration of structure and function of the ribosome actively engaged in protein synthesis, by cryo-electron microscopy (cryo-EM) and single-particle reconstruction. Protein biosynthesis is one of the most fundamental processes of life, but despite seminal work in solving the structure of the ribosome, this process is not well understood. Cryo-EM is uniquely suited to capture the molecule in functional states under close to native conditions. In this lab, several seminal discoveries regarding the dynamics of this process in both eubacterial and eukaryotic translation have been made in the past; among these the ratchet-like motion during mRNA-tRNA translocation and the large spring-like deformation of the aminoacyl-tRNA as it enters the ribosome during the aminoacyl-tRNA selection process. Special focus of this research program will be the eukaryotic (yeast and mammalian) ribosome. The aims of the proposed studies are threefold: (1) to make use of tilt pair recoding, novel technology for online data capture in the electron microscope, workflow optimization, and availability of a Titan Krios instrument at Janelia Farm, and other improvements in order to achieve atomic (better than 3.5A) resolution for selected ribosome complexes of special interest, such as ribosomes of trypanosomes and a yeast translocation complex; (2) to study the process of mammalian eukaryotic translation initiation by cryo-EM visualization of selected initiation complexes; (3) to study, with the aid of advanced classification methods, the distribution of states and their structural manifestations in a freely equilibrating, factor-free, eukaryotic ribosome sample as a function of relevant parameters such as temperature and ionic conditions. Samples will be obtained from several collaborating labs that are specialized in eukaryotic translation. Additional important collaborations are in the area of eukaryotic ribosome structure, smFRET, classification of heterogeneous samples by manifold embedding, and atomic modeling. Density maps when obtained at sufficient resolution will be analyzed by flexible fitting and interpreted in the rich context of existing structural, kinetics, single-molecule FRET, and biochemical data.